Intraocular liver spheroids for non-invasive high-resolution in vivo monitoring of liver cell function

Longitudinal monitoring of liver function in vivo is hindered by the lack of high-resolution non-invasive imaging techniques. Using the anterior chamber of the mouse eye as a transplantation site, we have established a platform for longitudinal in vivo imaging of liver spheroids at cellular resolution. Transplanted liver spheroids engraft on the iris, become vascularized and innervated, retain hepatocyte-specific and liver-like features and can be studied by in vivo confocal microscopy. Employing fluorescent probes administered intravenously or spheroids formed from reporter mice, we showcase the potential use of this platform for monitoring hepatocyte cell cycle activity, bile secretion and lipoprotein uptake. Moreover, we show that hepatic lipid accumulation during diet-induced hepatosteatosis is mirrored in intraocular in vivo grafts. Here, we show a new technology which provides a crucial and unique tool to study liver physiology and disease progression in pre-clinical and basic research.


REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): 1.The 3D culture system showed promising features such as increased expression of liver biomarkers and improved microvasculature structures, so it may serve as Intraocular liver spheroids for non-invasive high-resolution in vivo monitoring of liver cell function.However, the manuscript was poorly written, with the results not clearly presented and details of approaches missing.It raises concerns about the soundness of scientific interpretations and the conclusions.
2. Figure 1 lacks quantitative analysis and detailed explanation.The diameters of spheroids should be show.Also, liver spheroids should be showen with cell viability data.
3. For Figure 1, liver spheroid should be provided for hepatocytes characteristic and microstructure data.4. Figure 2 said In vivo imaging of labeled red blood cells (red) travelled through intraspheroid vessels but It is not easy to distinguish vessels in Fig 2 .It would be better to mark the blood vessels. 5.In figure 4, I wonder why none of glucose/lipid metabolism pathways were selected as they were so important for liver functions.Also, how the 10 genes were selected for the real-time PCR? 6.I think, Vascularization is achieved mainly with the presence of LSECs.Please add discussion or proof for liver spheroid or host induced vascularization.7. What is the size of liver spheroid in vitro or invivo?How do you measure spheroid size? 8. What causes blood vessels to grow in the eye? 9. What are the criterion for selecting media in PHS and LSECS mixed culture?

Dear Authors,
The manuscript is built on the extensive expertise and anterior chamber of the eye (ACE) models already developed by some of the authors.This provides already a good robust platform where to further develop the platform for longitudinal in vivo imaging of liver spheroids at cellular resolution.The conceptual development and need for a cellular resolution transplanted liver spheroids with demonstrated hepatocyte-specific and liver-like physiological functions is sound and clearly presented.
The imaging approach taken to show case the mature hepatocyte response is also confirmed visually, down at cell-cluster level but also by means of molecular assays showing similar cellular-and organ-response to human liver functions at both healthy and disease stage (e.g., hepatosteatosis).
Given the relevance that this platform model could provide in addressing the continuous screening of pharmacological drugs or possible toxins, it is important that all aspects associated with the development and implementation of this will be taken into account to avoid to fall short of information while running the exposure assessment.
Thus, some points need to be improved and presented as the platform model and its primary objectives are at the centre of the 3R ethical discussion.It is obvious that the platform offers a simultaneous multiple spheroids investigation and observation which bring it into the reduction and/or refinement on the need for animal use.This poses several requests for clarification which I would like the authors to take into account in the manuscript revision, since are not presented in the current version: -Animal welfare, husbandry, sacrifice and behaviour should be presented as part of the in vivo ethical justification for the study.Please also include ethical authorisation and specify if this was a single isolated study or part of a series of and for what intention this was authorised.
-For the animal behaviour the animals weight (e.g., loss or gain) should be presented as this is part of the animal study records.If blood chemistry was carried out it would be interesting to see the comparison between the study branches and the behaviour.
-For the two points above, the authors could also referenced and draw comparison with their previously developed ACE models, if they wish to include.
-For the in vivo study the injection and imaging setup is briefly described, I believe the readers would benefit from a visual image of the rig and also animal positioning, setting up time and total imaging lenght time.
-Regarding the upright laser scanning confocal microscope, it is known that ablation is occurring while the sample is imaged.The extent of this is proportional to the laser intensity, acquisition time, pinhole aperture and number of images taken.As the authors are also using different fluorescent dyes with different excitations and emission wavelengths, the technical information should be provided in order to assess if the damaged induced to the host-organ is reversible or irreversible.
-In light of the above point, based on the imaging settings, the upright laser scanning confocal microscope, there is no details on how the spheroids is impacted by every imaging session through to the culture time since these are grown up to 6-months.
-Clarification: please expand on the sentence:"... platform for non-invasive and longitudinal in vivo imaging of mouse liver spheroids in the ACE at cellular resolution…".
-Clarification: please expand on the selection of the cell lineages adopted in this study and future statistical robustness, as the variability among primary cell lines is often very high.
-With the continuous thrive that 3D biology is offering nowadays, I would also encourage the authors to a broader reflective approach on how the platform will compete against possible alternative platforms which are not as time and labour intensive as the one presented.
The authors have made quite a large number of statements in the final section of the discussion which are in need of some clarification.
Given the authors will address all the above points in details, I would be happy to reconsider the manuscript for acceptance and possible publication.

COMMENTS REVIEWER #1
We thank this reviewer for the consideration of our work and the valuable comments which we addressed in our response.
Comment 1: The 3D culture system showed promising features such as increased expression of liver biomarkers and improved microvasculature structures, so it may serve as Intraocular liver spheroids for non-invasive high-resolution in vivo monitoring of liver cell function.However, the manuscript was poorly written, with the results not clearly presented and details of approaches missing.It raises concerns about the soundness of scientific interpretations and the conclusions.

Response:
In reference to the writing of the manuscript, we have revised the main text to improve the clarity of the results and conclusions.Specifically, we have substantially edited the Results section related to the transplantation of human liver spheroids, as well as the concluding paragraph of the Discussion.We have moreover added explanations and details to the Methods section.We also included new supplementary information concerning aspects of mouse in vitro liver spheroid characterisation, the transplantation of human liver spheroids and in vivo imaging settings, which we explain in further detail in the following responses.

Response:
We have expanded Fig. 1 (by adding panels c and d) to contain the in vivo imaging set up and positioning of the mouse, as well as a timeline of in vivo imaging preparation and duration.We recognized we have missed the spheroid size information and we have now addressed it in the Supplementary Fig. 1.The average size of the in vitro spheroids selected for transplantation is of 248±13 µm (mean±SD), calculated by averaging the vertical and horizontal diameters of each spheroid imaged in the 96-well plate in which they are formed plating 1200 cells/well.Our reasons for selecting spheroids of this size are the following: (1) the spheroid size should not be too large to avoid hypoxia and necrotic core, but they should contain enough cells to support cellcell communications and to allow graft remodelling in the eye, (2) the weight of spheroids of this size allows them to gravitate towards the iris and allow their engraftment, (3) this size is appropriate in relation to transplanting 5-10 spheroids per mouse eye.As to the size of liver spheroids engrafted in the eye post-transplantation, we have calculated the size of the spheroids by in vivo imaging, at 3 weeks post-transplantation, when we consider the liver spheroids have reached a stable shape and size.In the in vivo situation, we also calculated their size by averaging the vertical and horizontal diameters.We have included these data and the method used, along with a schematic on how we calculate spheroid size both in vitro and in vivo, in Supplementary Fig. 1e-g.
Regarding the cell viability of in vitro liver spheroids prior to transplantation, we agree that this aspect was not addressed.Therefore, we have performed a live/dead cell assay on liver spheroids in culture to determine their cell viability.The spheroids showed high cell viability (99.2±0.4 %; mean±SD) with very few dead cells mainly located to the spheroid surface and no evidence of necrotic core.We have added this new data into Supplementary Fig. 1h.Regarding the cell viability of the liver cells within the grafts in vivo, we directly demonstrate the functioning of the hepatocytes through the diverse characterisation experiments in the manuscript (e.g.bile export and lipoprotein uptake in Fig. 3-4), which show that the cells are viable and perform their parenchymal tasks.
Response: Liver spheroids have been widely used over the past decade as a 3D liver in vitro tissue model, with many studies that provide in-depth characterisation of this model [1,2]: In response to this comment, we added a characterisation of our cultured liver spheroids prior to transplantation.Therefore, we have added RNA in situ hybridization, showing single-cell spatial gene expression of hepatic markers Albumin and HNF4a (Supplementary Fig. 1a), as well as immunostaining of structural proteins F-actin (phalloidin) and E-cadherin (CDH1) (Supplementary Fig. 1b,c).Moreover, we point out typical bi-nucleated hepatocytes within the spheroid (Supplementary Fig. 1d).After transplantation and engraftment, the liver spheroids have been extensively characterised in the eye throughout the paper, showing the expression of hepatocyte-specific markers (Fig. 3a,b,c), evidence of hepatocyte cell microstructure (Fig. 3d) and proof of hepatocyte functionality by in vivo assays (Fig. 4).

Response:
We thank the reviewer for this comment and have made changes to make this experiment clearer.Namely, we have substituted this panel for new representative images of an experiment in which we co-injected the labelled RBCs with lectin, which stains the blood vessels in vivo (Fig. 2f).Accordingly, we have also substituted Supplementary Movie 1 with this new experiment.
In our Figure 4 (b,f), we selected those 10 genes mined from the bulk RNA sequencing dataset due to their connection to the imaging experiment shown in the same figure (bile acid assay and LDL uptake).We selected those representative genes, which are known to be key players in bile and lipid metabolism from publications focussing on the characterisation of hepatocytes [2][3][4].
Regarding the lack of gene expression for glucose/lipid metabolism pathways, in Supplementary Fig. 4b we show gene expression data related to glucose and glycogen metabolism, while in Fig. 4f, we show key lipid metabolism genes supported by an in vivo assay of LDL-uptake in hepatocytes (Fig. 4g).However, in the main figures, glucose metabolism related genes were not added since our main goal was to show the imaging capabilities of our platform related to different hepatocyte functions.Since we could not find specific probes/assays which can measure this specific aspect in vivo, we added a few representative genes derived from the mining of our RNAseq dataset in the Supplementary Fig. 4.

Response:
We have revised the section of the manuscript on the transplantation of human liver spheroids into recipient immunocompromised mice which, as pointed out by the reviewer, lacked the explanation of the rationale behind the use of primary human liver sinusoidal endothelial cells (LSECs).When transplanting liver spheroids made of purified primary human hepatocytes (PHH), these spheroids failed to engraft and become vascularised in the mouse eye.We attribute this to the purchased PHH being highly purified and therefore lacking supporting cell types, such as endothelial cells or Kupffer cells, both of which are known to secrete pro-angiogenic and growth factors, which promote vascularisation [5,6].Additionally, there could be species-specific angiocrine signals which are hindered by the transplantation of human tissue into mouse recipient.Therefore, we hypothesized that the addition of primary human LSECs could facilitate the vascularisation of the graft.This strategy has been used by others, for example, in a study by Takebe et al. [7], iPSC-derived hepatocytes were cultured with supportive mesenchymal and endothelial cells to form 3D-liver buds, which were then transplanted onto the mouse brain, where they successfully engrafted and became vascularised.Thus, we generated liver spheroids from a 2:1 mixture of PHH and LSECs in vitro and transplanted them into the ACE, where they became vascularised.Therefore, we agree with Reviewer #1, that the vascularisation of human liver spheroids in the ACE of mice is made possible by the presence of LSECs and we have added this in the Results (Page 5 line 3-12) and Discussion (Page 10 line 13-28).

Response:
The average size of liver spheroids in culture prior to transplantation is of approx.250 µm, corresponding to seeding 1200 cells/well and the size is measured by calculating the average of vertical and horizontal diameters.Upon transplantation into the ACE, the spheroid mass can be easily differentiated from the iris tissue due to different backscatter signal intensity, therefore we use the same strategy to measure spheroid size.We have added this new data into the supplementary material, alongside a schematic explanation of how the spheroid size was calculated (Supplementary Fig. 1e-g).
Response: For the co-culture of PHH and LSECs, we followed the protocols by Ware et al. and Bale et al, in which primary hepatocytes and LSECs are co-cultured [12,13].The composition of the Wiliams E hepatocyte media we used is optimized to avoid the de-differentiation of primary hepatocytes in culture.In response to this comment, we also provide new immunofluorescence staining of CD31-positive endothelial cells within the spheroid mass (Supplementary Fig. 2d), showing that LSECs survive and are present in the co-culture spheroids prior to transplantation.

General response to Reviewer #2:
We appreciate the positive and constructive criticism from Reviewer #2 and we are confident we provided the additional data and information the referee requested.
Comment 1: Animal welfare, husbandry, sacrifice and behaviour should be presented as part of the in vivo ethical justification for the study.Please also include ethical authorisation and specify if this was a single isolated study or part of a series of and for what intention this was authorised.

Response:
We agree and modified the first section in the Methods as "Animal welfare, husbandry, behavior and sacrifice" and added an "Ethical authorization" paragraph.
Firstly, we now explain that mice were housed in littermate groups of 2-6 animals and importantly, that there was no need for animals to be in isolation neither prior nor after surgery or in vivo imaging sessions.We believe this aspect is crucial for animal welfare, given that mice are social animals.We have also added details on the sacrifice of the animals, which is carried out at the end of the experiment but is not determined by health deterioration of the animal caused by the transplantation surgery or in vivo imaging sessions.Additionally, we now reiterate in the Methods section that the operated animals recover quickly from the surgical intervention, with no need of post-operative care, and that the transplantation does not affect their behaviour.We consider normal behaviour to be aspects such as, no increased fighting between males, normal feeding and growth, normal levels of activity and nest-making.Moreover, transplanted animals have been maintained for over 1 year, with no apparent effect to their health or longevity.
Secondly, we cover the ethical authorisation.This study is covered by our group's ethical permit "Studies on the function of hormone-releasing and hormone-stimulated cells and related cells/tissues in normal and diabetic animal models and in transplanted tissues and cells" (n.6362-2023, previous 16454-2022, 17431-2021, 8822-2020) approved by the Animal Experiment Ethics Committee at Karolinska Institutet.This permission describes the transplantation of microtissues into the ACE, with the objective of establishing platforms for non-invasive in vivo imaging of tissues at cellular resolution.The ethical justification for these types of studies is because, beside the research purposes, longitudinal imaging of the same cells in the same subject over time allows the reduction of experimental animals, while at the same time improving the quality of the in vivo data, given that we can monitor reaction and progression in individual cells over time.Thus, this study forms part of a larger effort to promote the anterior chamber of the eye as a platform for non-invasive imaging of different tissues and its application in both basic and pre-clinical medical research.
Response: Given that our group has longstanding experience with the implementation of this imaging method, we did not originally record the weight progression of transplanted vs nontransplanted animals.However, we agree that it is important to show the effects of the transplantation surgery and in vivo imaging in recipient mice.In addition to recording weight gain/loss, we have monitored non-fasting blood glucose levels in both groups, as an indirect measure of sustained stress and metabolic alterations.We found that neither weight nor blood glucose levels significantly differed between the transplanted and non-transplanted groups over a period of 1-month post-transplantation. Additionally, we performed in vivo imaging at 3 weeks post-transplantation in recipient animals, which did not have a negative effect on their health.We have created Supplementary Fig. 6. to include these new data.

Response:
We have added an image showing the in vivo imaging set up and positioning of the mouse (Fig. 1c), as well as a timeline (Fig. 1d) depicting the set-up steps and imaging duration.

Response:
We have created Supplementary Table 2, in which we summarize the settings we use for in vivo imaging of the different fluorescent reporter proteins and injectable fluorescent probes.
Response: Despite repeated imaging sessions, the laser exposure time per spheroid is limited to 45-60 seconds, in case of capturing a 3D spheroid Z-stack scan (at 600 Hz speed and stacks of 4 µm thickness).If capturing individual images of clusters of cells, a single plane image is taken in less than one second.We believe the impact of laser incidence on the spheroids is negligible, for the following reasons.On the macroscopic level, we see no changes to the appearance of the iris and cornea after the imaging sessions (no signs of redness, irritation or cloudiness of the aqueous humour).In terms of animal welfare and pain, the mice show no signs of pain when awakening from an in vivo imaging session (according to the Mouse Grimace Scale; score of 0 after 30 min of recovery).On the microscopic level, the imaging does not induce photobleaching (given we use the same laser power to observe a given fluorophore in repeated imaging sessions), nor do we see any damage to cells or organelle structures, which show intact functionality despite having been imaged multiple times.Moreover, as Reviewer #2 has alluded to, our research group relies on over a decade of experience with this imaging platform to conclude that longitudinal in vivo imaging sessions, if performed under our conditions, do not cause photodamage or have a detrimental effect on the transplanted tissue or the recipient animal.To clarify this in the text, we have added a sentence in the Methods section (Page 15 lines 28-29) to explain that photodamage does not occur in the imaged liver spheroids when using our imaging conditions.

Response:
We have rephrased the sentence to more clearly highlight the two main advantages of this imaging platform; the possibility of longitudinal studies and high resolution imaging at singlecell level.
Original sentence: In conclusion, we have established and characterized a platform for noninvasive and longitudinal in vivo imaging of mouse liver spheroids in the ACE at cellular resolution.
Revised version: In conclusion, we have established and characterized a platform for in vivo imaging of mouse liver spheroids, in which the graft can be imaged non-invasively and repeatedly at different time points in the same animal (longitudinally) at single-cell resolution.

Response:
We apologize for the confusion.In this study we did not use any cell lines but only primary cells, isolated directly from adult mouse or human liver.Notably, these primary liver cells are not proliferative.To avoid or reduce inter-donor variability of primary cells (and thus transplanted spheroids) in our experiments, we isolated primary cells from animals of the same strain and selected similar age and sex.In the case of human material, we agree that inter-donor variability can be high.However, as cells can be cryopreserved, it is possible to use material from the same donor in multiple experiments, which allows to abstract from inter-individual variability.

Response:
The ACE is a unique transplantation site in terms of its optical accessibility, and compared to the installation of an abdominal body window [14], the ACE surgery is notably quicker, easier and less invasive for the animal.Therefore, we have not previously commented on this aspect in our Discussion.However, we are not imaging the endogenous liver, and it should not be considered as another form of liver intravital imaging.We do not envision our model as replacement of in vitro 3D liver models.Rather, we believe that our platform would be used in combination to high-throughput in vitro techniques.For example, as a platform for in vivo validation of compounds or therapies, which have previously been explored by in vitro studies.We have added this last consideration to the Discussion (Page 11 lines 4-7).
Response: Thanks for the valuable suggestion.We have revised as follows.
Original paragraph: In conclusion, we have established and characterized a platform for noninvasive and longitudinal in vivo imaging of mouse liver spheroids in the ACE at cellular resolution.These spheroids preserve liver-like features and wherein hepatocytes retain their overall differentiation and functionality.By different proof-of-concepts experiments we have shown the capabilities of this platform spanning multiple areas of liver research and we foresee a future in both basic research and as a testing-tool for therapeutics in pre-clinical and translational studies.
Revised version: In conclusion, we have established and characterized a platform for in vivo imaging of mouse liver spheroids, in which the graft can be imaged non-invasively and repeatedly at different time points (longitudinally) in the same animal at single-cell resolution.The liver spheroids preserve liver-like features and retain hepatic differentiation and functionality.By different proof-of-concepts experiments, we have shown the monitoring capabilities of this platform in different areas of liver research, such as metabolic disease and liver regeneration.We believe this platform will be of great value in both basic research and translational studies.